Quantitative Phosphoproteomic Analysis Reveals Potential Regulatory Mechanisms of Early Fruit Enlargement in Pecan (Carya illinoinensis)

Pecan
(Carya illinoinensis) is a
popular tree nut. Its fruit development undergoes slow growth, rapid
expansion, core hardening, and kernel maturation stages. However,
little is known about how pecan initiates fruit development and enlargement
after pollination. In this study, we performed the first large-scale
identification of potential phosphorylation sites and proteins at
early development of pecan fruit by a label-free phosphoproteomic
quantification technique. A total of 2155 phosphosites were identified
from 1953 phosphopeptides covering 1311 phosphoproteins in unpollinated
pistils and fruits at 5 and 9 weeks after pollination. Of these, 699
nonredundant phosphoproteins were differentially phosphorylated (DP).
Furthermore, the phosphorylation intensity of DP proteins in brassinolide
(BR) and auxin signaling were analyzed, and the function of CiBZR1
was investigated. Ectopic expression of CiBZR1 resulted
in BR response phenotypes with curled leaves and fruit, while enlarged
seed size in Arabidopsis. Subcellular
localization and transcriptional activation activity assay demonstrated
that CiBZR1 distributed in both the nucleus and cytoplasm with transcriptional
activity. When two phosphosites mutated, CiBZR1S201P,S205G moved to the nucleus completely, while the transcriptional activity
remained unchanged. Taken together, our data reveal extensive phosphoproteins
and lay a foundation to comprehensively dissect the potential post-translational
regulation mechanism of early development of pecan fruit.